Peptide mapping with mass spectroscopy (“MS”) detection enjoys widespread use in protein analytics, particularly for confirmation of the primary sequences of many types of proteins, including MAbs. However, one limitation of existing MS analytical methods is that they are primarily focused on qualitatively confirming the presence of expected peptides.
A recent development that allows for the extension of MS detection to quantitative analysis of protein samples is the availability of isotopically-labeled amino acids. For example, proteins comprising isotopically-labeled amino acids can be utilized in the Stable Isotope Labeling with amino acids in Cell culture (“SILAC”) method to detect differences in protein abundance between two or more samples.
The SILAC method involves first establishing two independent cell cultures. The first culture is supplemented with isotopically-labeled amino acids and the second is supplemented with standard amino acids. After sufficient time has elapsed to allow for uniform incorporation of the isotopically-labeled amino acids into the proteome of the first culture (e.g., 5 cell doublings), the proteomes of the two cultures (or sub-proteomes thereof) are isolated, digested, and subjected to MS analysis. If a protein of interest is expressed in both the isotopically-labeled culture and the standard culture, the digestion step will result in peptide fragment pairs where one member is isotopically labeled and one that is not. Because MS analysis results in an intensity peak for each peptide fragment of a digested protein, each peptide fragment pair will appear as two distinct MS intensity peaks, which are separated by the mass of the isotopic label(s) incorporated into the labeled member of the pair. The two MS intensity peaks for any one peptide fragment pair can be compared and the ratio of the peak intensities reflects not only the presence of the protein in each of the two cultures, but also allows for the calculation of an abundance ratio for the proteins made by the two cultures. Thus, the SILAC method can be used to qualitatively confirm the presence of particular proteins in a cell population and to quantitatively determine protein abundances in different cell populations.
Although the SILAC method allows for certain types of qualitative and quantitative analysis, there remains a need in the art for protein analytics capable of qualitatively and/or quantitatively detecting the presence of (1) mutations, e.g., insertions, deletions, or substitutions; (2) modifications, e.g., the presence of glycosylation or methylation; or (3) polypeptide impurities, e.g., polypeptides present in a sample that have one or more identical sequence fragments when compared to a protein of interest, including, but not limited to a degradation product of the protein of interest. The instant invention addresses this need.